Neurotrophic factors are natural proteins, found in the nervous system or in non-nerve tissues innervated by the nervous system, whose function is to promote the survival and maintain the phenotypic differentiation of nerve and/or glial cells (Varon and Bunge 1978 Ann. Rev. Neuroscience 1:327; Thoenen and Edgar 1985 Science 229:238). Because of this physiological role, neurotrophic factors may be useful in treating the degeneration of nerve cells and loss of differentiated function that occurs in a variety of neurodegenerative diseases, such as Alzheimer's or Parkinson's diseases, or after traumatic injuries, such as stroke or physical trauma to the spinal cord (Appel 1981 Ann. Neurology 10:499).
In order for a particular neurotrophic factor to be potentially useful in treating nerve damage, the class or classes of damaged nerve cells must be responsive to the factor. Different neurotrophic factors typically affect distinctly different classes of nerve cells. Therefore, it is advisable to have on hand a variety of different neurotrophic factors to treat each of the classes of damaged neurons that may occur with different forms of disease or injury.
A given neurotrophic factor, in addition to having the correct neuronal specificity, must be available in sufficient quantity to be used as a pharmaceutical treatment. Also, since neurotrophic factors are proteins, it would be desirable to administer to human patients only the human form of the protein, to avoid an immunological response to a foreign protein.
Since neurotrophic factors are typically present in vanishingly small amounts in tissues (e.g., Hofer and Barde 1988 Nature 331:261; Lin et al. 1989 Science 246:1023) and since human tissues are not readily available for extraction, it would be inconvenient to prepare pharmaceutical quantities of human neurotrophic factors directly from human tissues. As an alternative, it would be desirable to isolate the human gene for a neurotrophic factor and use that gene as the basis for establishing a recombinant expression system to produce potentially unlimited amounts of the human protein.
Two neurotrophic factors have been described that are closely related in amino acid sequence but which affect different, although partially overlapping, sets of responsive neurons (Leibrock et al. 1989 Nature 341:149). These two neurotrophic factors are: (1) nerve growth factor (NGF) and (2) brain-derived neurotrophic factor (BDNF). Both NGF and BDNF are apparently synthesized as larger precursor forms which are then processed, by proteolytic cleavages, to produce the mature neurotrophic factor (Edwards et al, 1986 Nature 319:784; Leibrock et al. 1989 ibid.). The only genes for members of the proposed NGF/BDNF family of neurotrophic proteins that have been reported to date are the human and various animal genes for NGF (Scott et al. 1983 Nature 302:538; Ullrich et al. 1983 Nature 303:821; Meier et al. 1986 EMBO J. 5:1489) and the pig gene for BDNF (Leibrock et al. 1989 ibid.). There is a significant similarity in amino acid sequences between mature NGFs and mature BDNF, including the relative position of all six cysteine amino acid residues, which is identical in mature NGFs and BDNF from all species examined (Leibrock et al 1989 ibid.). See FIG. 7, comparing and emphasizing the similarities of human forms of NGF and BDNF. This suggests that the three-dimensional structure of these two proteins, as determined by the location of disulfide bonds, is similar. Both mature proteins also share a basic isoelectric point (pI).
NGF is a neurotrophic factor at least for cholinergic neurons in the basal forebrain (Hefti and Will 1987 J. Neural Transm. [Suppl] (AUSTRIA) 24:309). The functional inactivation and degeneration of the basal forebrain cholinergic neurons responsive to NGF in the course of Alzheimer's disease is thought to be the proximate cause of the cognitive and memory deficits associated with that disease (Hefti and Will 1987 ibid.). NGF has been shown to prevent the degeneration and restore the function of basal forebrain cholinergic neurons in animal models related to Alzheimer's disease, and on this basis has been proposed as a treatment to prevent the degeneration and restore the function of these neurons in Alzheimer's disease (Williams et al. 1986 Proc. Natl. Acad. Sci. USA 83:9231; Hefti 1986 J. Neuroscience 6:2155; Kromer 1987 Science 235:214; Fischer et al. 1987 Nature 329:65).
BDNF is a neurotrophic factor for sensory neurons in the peripheral neurons system (Barde 1989 Neuron 2:1525). On this basis, BDNF may prove useful for the treatment of the loss of sensation associated with damage to sensory nerve cells that occurs in various peripheral neuropathies (Schaumberg et al., 1983 "Disorders of Peripheral Nerves" F. A. Davis Co., Philadelphia, Pa.).
Recombinant expression systems that are capable of producing the large quantities of fully-biologically-active and structurally-unmodified mature NGF needed for pharmaceutical development are highly desireable. See, European Patent Publication EP 89113709, describing the recombinant expression of NGF in insect cells. Mature, biologically-active, NGF can be produced when human or animal NGF genes are expressed in eukaryotic cell expression systems (e.g., Edwards et al. 1988 Molec. Cell. Biol. 8:2456). In such systems, the full-length NGF precursor is first synthesized and then proteolytically processed to produce mature NGF which is correctly folded 3-dimensionally and is fully biologically active. However, eukaryotic cell expression systems often produce relatively low yields of protein per gram of cells and are relatively expensive to use in manufacturing.
In contrast, expression systems that use prokaryotic cells, such as bacteria, generally yield relatively large amounts of expressed protein per gram of cells and are relatively inexpensive to use in manufacturing. However, an adequate bacterial expression system capable of producing fully-biologically-active and structurally-unmodified mature NGF has not been described. A bacterial expression system is disclosed in Canadian Patent No. 1,220,736. However, no procedures for refolding the expressed protein are presented. This failure can probably be traced to problems associated with bacterial expression systems in general and problems associated with the specific techniques employed to produce NGF in bacteria.
Bacteria are not able to correctly process precursor proteins, such as the precursor protein for NGF, by making appropriate proteolytic cleavages in order to produce the correct smaller mature protein. Therefore, to produce mature NGF in bacteria, it is necessary to express only that portion of the NGF DNA sequence encoding the mature protein and not that for the larger precursor form. When this was done in the bacterium Escherichia coli, relatively large amounts of the mature human NGF protein were produced (see, e.g., Iwai et al. 1986 Chem. Pharm. Bull. 34:4724; Dicou et al. 1989 J Neurosci. Res. 22:13; EP application 121,338). Unfortunately, the bacterially-expressed protein had little or no biological activity.
A protocol for refolding bacterially expressed NGF has been described in European Patent Application 336,324 which restores some biological activity to mature NGF produced in bacteria. However, this protocol has serious deficiencies.
Mature human NGF has generally been unavailable in sufficient amounts for pharmaceutical use, since many eukaryotic expression systems are expensive and often do not produce adequate amounts of mature NGF. Bacterial expression systems described so far have not produced biologically-active and chemically-unmodified mature NGF in sufficient quantities for pharmaceutical use. Since human mature NGF is likely to be useful in the treatment of Alzheimer's disease, the unavailability of this material has been keenly felt by the scientific and clinical communities. The unavailability of biologically-active human mature NGF was seen by a panel of leading scientists, assembled by the National Institute on Aging, as the critical block to further development of NGF as a treatment for Alzheimer's disease (Phelps et al. 1989 Science 243:11).
It is presumed that similar manufacturing difficulties would apply to each member of the NGF/BDNF family of neurotrophic proteins, since members of this family so far described have identically located cysteine amino acid residues and presumably, therefore, form a pattern of intramolecular disulfide bonds identical to that of NGF (Angeletti et al. 1973 Biochemistry 12:100).
In view of the apparent value of such neurotrophic proteins and the current restraints on the production of large quantities of the biologically active proteins as indicated above, it would be desirable to provide the following: (1) the identification, isolation and characterization of all members of the NGF/BDNF family of neurotrophic proteins; i.e., proteins that are structurally related to NGF and BDNF in a manner similar to the way these two proteins are related to each other; (2) the identification, isolation and characterization of all naturally occurring human members of the NGF/BDNF family of neurotrophic proteins, including specifically human BDNF; (3) the isolation and characterization of genes coding for any and all members of the NGF/BDNF family of neurotrophic proteins, including specifically the human genes coding for all such family members; (4) methods for using the human genes to establish recombinant expression systems in microorganisms such as E. coli that will produce significant quantities of the mature (processed) form of these human proteins; (5) methods for refolding members of the NGF/BDNF family of neurotrophic proteins to allow them to obtain a biological specific activity; and (6) pharmaceutical compositions for the treatment of neurological diseases comprised of any one or any combination of the members of the NGF/BDNF family of neurotrophic proteins.